EpiGenie | Epigenetics, Stem Cell, and Synthetic Biology Newshttp://epigenie.com
Scientific News, Technology, and Product InformationMon, 02 Mar 2015 09:24:02 +0000en-UShourly1http://wordpress.org/?v=4.1.1Epigenetic Sequencing Woes Overcome by Services Know-howhttp://epigenie.com/epigenetic-sequencing-woes-overcome-by-services-know-how/
http://epigenie.com/epigenetic-sequencing-woes-overcome-by-services-know-how/#respondSun, 01 Mar 2015 21:31:39 +0000http://epigenie.com/?p=22241Sequencing technologies have created a dizzying number of opportunities for epigenetic researchers to explore questions that couldn’t be tackled before. Despite the immense power that sequencing delivers, though, the whole process still isn’t quite as simple as pushing a big red button, and watching the groundbreaking discoveries roll in. Even as sequencing becomes faster, easier, and less expensive those experienced in the field have realized that the work doesn’t end once the run is finished.

We recently caught up with Dr. Keith Booher, who clued us into some of the things that the Epigenetics Sequencing Group at Zymo Research has learned from their extensive experience running NGS studies for both clients and internal R&D teams, and how it’s shaped the services that they offer.

Bioinformatics – Insights, Not Just Data

One of the first things that the Zymo crew recognized from early their NGS work, was that researchers really need more than just a hard-drive filled with terabytes of data from their sequencing service; they need help processing that information, too.

Data analysis and bioinformatics is crucial to making sense out of the complex data that sequencing generates, but while epigenetics researchers have a keen understanding of biological mechanisms, they may not always have the time or specialized resources to translate large data sets.

“Our services clients wanted insights, not just data. So, we began to offer up our bioinformatics expertise to create a way for researchers to bypass the data crunching, and focus their efforts on scientific discovery.” said Booher.

These days that couldn’t be easier – just send in samples, and the Zymo Bioinformatics Team will provide a customizable, comprehensive, genome-wide analyses report complete with publication-ready graphs and figures.

DNA Methylation Analysis Isn’t One-size-fits-all

Sure, it would be wonderful to sequence an entire genome for every tissue, in every organism, under every condition. But, is that really the most efficient approach?

Zymo employs a variety of DNA methylation sequencing methodologies for their services, and has found that when used in combination, they can be a streamlined and cost-effective way to zero-in on exactly the data you need.

The experts at Zymo Research have figured out that selecting the right technique, or combination of techniques, enables scientists to get the most bang for their sequencing buck.

Integrate Those Epigenetic Mechanisms

If the advanced sequencing revolution has proven anything, it’s that genomic regulation is way more complex than originally predicted. While early epigenetic studies generally focused on one type of epigenetic mechanism, recent investigations have highlighted the need to look at epigenetic regulation from multiple angles, and today’s high-powered sequencing methods have made this possible.

The flexibility to take an integrated snapshot of the epigenome from multiple angles at once makes the Zymo Research suite of services an attractive option for researchers in need of comprehensive data, but without the resources to conduct multiple analyses simultaneously.

To take advantage of the hard won knowledge and expertise of the Zymo crew, check out their Epigenetics Services page, to see available options.

]]>http://epigenie.com/epigenetic-sequencing-woes-overcome-by-services-know-how/feed/0Express Yourself with Light-activatable CRISPR-Cas9http://epigenie.com/express-yourself-with-light-activatable-crispr-cas9/
http://epigenie.com/express-yourself-with-light-activatable-crispr-cas9/#respondThu, 26 Feb 2015 09:39:19 +0000http://epigenie.com/?p=22197Guess what? It seems that blue light has a lot more to offer than just helping with your winter time blues. It could also be just what your transcriptional activation system needs. Synthetic biology has a lot to offer omics beyond genome editing and recent work from multiple groups is putting Cas9 in a different spotlight.

This neat little system comes at you right from Arabidopsis, with a duo of protiens:

Light-sensitive cryptochrome 2 (CRY2).

Binding partner CIB1.

The binding, which leads to their heterodimerization, is rapid and reversible, you’ve just gotta shine a little blue light.

In the LITE system, the TALE DNA binding domain was fused to CRY2 (thus giving sequence specificity by protein binding to DNA) and the VP64 activator was fused to CIB1. Then when CRY2 gets a little over excited it binds to CIB1 and drives expression.

Light-Activated CRISPR-​Cas9 Effectors

This system has now been tweaked to use CRISPR/Cas9 as shown in two recent papers. By using a combination of optogenetics and CRISPR/Cas9, the teams were able to have their systems on call for some spatio-temporal control.

The CRISPR version goes by the flashy name of LACE (light-activated CRISPR-​Cas9 effector) or LITE 2.0, depending on the pub. It also has a few more improvements than just swapping out the TALE for dCas9 and was shown off in multiple chosen endogenous mammalian genes. The two developed systems only appear to differ by the CRY2 domains per a dCas9 and also the activator of choice. Ultimately, optogenetics provides the perfect ‘on’ switch for an on-the-fly fusion of dCas9 and a transactivation domain that gets the expression going. By using the dCas9 fusion the teams were able to take advantage of the excellent sgRNA targetting system that has an almost infinite number targets and is much cheaper than engineering targeting proteins, all the while not chopping away at the gene of interest. We’re left wondering how much longer until one of our favorite epigenetic modifiers joins the light show.

]]>http://epigenie.com/express-yourself-with-light-activatable-crispr-cas9/feed/0m6Ajor New Function for MicroRNAs in RNA Methylationhttp://epigenie.com/m6ajor-new-function-for-micrornas-in-rna-methylation/
http://epigenie.com/m6ajor-new-function-for-micrornas-in-rna-methylation/#respondFri, 20 Feb 2015 14:43:53 +0000http://epigenie.com/?p=22184It’s no secret that microRNAs are pretty busy molecules. A single microRNA can regulate the expression of several, maybe even hundreds, of transcripts. If that wasn’t enough, researchers at the Chinese Academy of Sciences, Beijing now add another function to this packed list of duties: the control of m6A RNA methylation, which is emerging as an important regulator of cell fate and differentiation.

If you are an epigenetics enthusiast you likely have already heard about m6A RNA methylation. Much like DNA methylation, m6A has enzymes that establish it (METTL3 and METTL14) and erase it (FTO and ALKBH5) on mRNA. This modification has been implicated in just about every aspect of mRNA metabolism. However, we still know surprisingly little about its importance on a global scale.

All this is about to change though with recent improvements to m6A-seq technology, which are enabling researchers to explore the landscape of m6A in different cell types. This technology has already been put to good use in embryonic stem cells, to reveal an important role for m6A in the control of cell fate decisions. Now, Qi Zhou and his team have created transcriptome-wide maps of m6A in four different cell types at various stages of differentiation.

Among other things, Zhou and colleagues used these new maps to answer a burning question: how is m6A regulated? After some careful data mining and some clever gain- and loss-of-function experiments they soon had their answer:

A staggering 92–96% of sequences enriched in m6A (‘m6A peaks’) overlapped with binding sites for microRNAs.

Overexpression of microRNAs pairing with randomly selected m6A peak regions led the accumulation of m6A at these regions.

Mutation of microRNAs created new binding regions on transcripts previously devoid of m6A peaks.

Thus, microRNAs appear to control m6A methylation. By tinkering with the expression of microRNAs through the knockdown of Dicer or the use of antagomirs, the team even manage to show how this is achieved: microRNAs control the interaction between METTL3 and mRNA.

Going further, they examine the function of m6A in cell fate decisions and find that METTL3 overexpression in mouse embryonic fibroblasts promotes the accumulation of m6A and improves the efficiency of induced reprogramming to pluripotency.

]]>http://epigenie.com/m6ajor-new-function-for-micrornas-in-rna-methylation/feed/0Getting started with Chromatin Conformation Capture (3C)http://epigenie.com/getting-started-with-chromatin-conformation-capture-3c/
http://epigenie.com/getting-started-with-chromatin-conformation-capture-3c/#respondMon, 16 Feb 2015 06:11:37 +0000http://epigenie.com/?p=22167The concept of chromatin contact mapping, or determining the three-dimensional structure conformation and interactions of chromatin domains, is now a reality because of Chromatin Conformation Capture (3C) and subsequent methods born out of that approach. Several new 3C-based techniques have emerged, each with particular strengths and applications, but the sheer variety creates a challenge when selecting the best method for a specific situation.

This guide from the good folks at Abcam will summarize current 3C methods, help you to choose the best option, and provide expert tips on optimizing the process in your lab. The guide covers:

]]>http://epigenie.com/getting-started-with-chromatin-conformation-capture-3c/feed/0New Transcriptional Repressors for Pick ‘N’ Mix Synthetic Gene Circuitshttp://epigenie.com/new-transcriptional-repressors-for-pick-n-mix-synthetic-gene-circuits/
http://epigenie.com/new-transcriptional-repressors-for-pick-n-mix-synthetic-gene-circuits/#respondMon, 16 Feb 2015 05:52:54 +0000http://epigenie.com/?p=22162As a kid there was no greater feeling on a weekend shopping trip than being set loose on the Pick ‘N’ Mix, carefully choosing from the wealth of candy to create the perfect selection. Lucky for you synthetic biology promises the same thrill, by providing a selection of ready-to-roll interchangeable parts to choose from allowing you to build complex synthetic gene circuits. One sticking point has held up this Pick ‘N’ Mix approach: the lack of well-characterized and orthogonal transcriptional repressors.

You may be familiar with classical synthetic transcriptional repressors, which typically involve the fusion of transcriptional repression domain to an engineered DNA binding protein domain, such as a Zinc finger protein, transcription activator-like effector (TALE) protein or deactivated Cas9 nuclease in a CRISPR system. The use of the transcriptional repressor domain has its problems, including that they tend to cause epigenetic modifications on the target promoter region, thereby interfering with temporal response due to delayed transcriptional reactivation.

To overcome this shortcoming, Li and colleagues decided to harness the technology that employs steric hindrance rather than repression domains. Specifically, the team constructed cell-type specific, microRNA-controlled transcription activator-like effector repressor (TALER) promoters that are transcriptionally inactivated upon TALER binding.

The TALER promoter consists of five upstream activation binding sites and a minimal cytomegalovirus (CMV) promoter flanked by two TALER binding sites. Mechanistically, constitutively expressed GalVP16 interacts with the upstream binding site and elicits the activation of the gene encoding the red fluorescence reporter mKate2. If a TALER protein is present, the repressor binds its 5’ and 3’ recognition sites situated at the ends of the minimal CMV promoter and thus suppressing transcription.

16 of the 23 TALERs blocked their target promoters by 100-fold more than the controls.

RNA-seq analysis revealed that TALERs are highly specific for their intended targets.

Having both the 5’ and 3’ binding sites available for TALERs and positioning TALER binding sites closer to the minimal CMV promoter increases the level of repression.

The authors concluded that the library of synthetic TALER circuit modules will have far-reaching biomedical and biotechnological applications, such as “precise cell-type classification for a mixed-cell population and sensing multi-input cell-type specific microRNA profile”.

Chromatin immunoprecipitation (ChIP) is the basis for much of modern chromatin analysis technologies. The central principle of ChIP is the use of an antibody targeting a protein or histone modification of interest to pull down DNA regions with which it associates. The adaptability and versatility of ChIP allow it to be applied to diverse research questions. This versatility is twofold: diversity of antibody choice and diversity of technology (qPCR, microarray, sequencing etc.).

Coupling of ChIP to microarray (ChIP-chip) has been pivotal to our understanding of the human genome. ChIP-seq has been instrumental for the ENCODE project which has mapped much more of the human genome. Furthermore, innovation of data analysis techniques can allow reanalysis or integration of data sets. This allows for new and often more reliable information to be uncovered.

Traditional ChIP has acted as a springboard to develop novel adaptations, which often combine ChIP with other techniques to address diverse biological problems. Restriction digestion of cross-linked chromatin allows for ligation of interacting fragments. This can be used to determine the three-dimensional organization of the genome. ChIP can also be combined with epigenetic technologies such as bisulfite conversion to interrogate the interplay between histone modification and DNA methylation.

This guide from our colleagues at Abcam, outlines the latest advanced ChIP-based techniques, and describes how their application has enabled new understanding of epigenetic mechanisms.

]]>http://epigenie.com/chip-2-0-guide-to-advanced-chromatin-immunoprecipitation-techniques/feed/0Plant Receptor Re-engineered to Hear “Drought!” From a New Signalhttp://epigenie.com/plant-receptor-re-engineered-to-hear-drought-from-a-new-signal/
http://epigenie.com/plant-receptor-re-engineered-to-hear-drought-from-a-new-signal/#respondThu, 12 Feb 2015 22:45:41 +0000http://epigenie.com/?p=22152Don’t get me wrong, plants have a lot of things going for them. However, as Groot’s fellow Galaxy Guardians can attest, vocabulary isn’t one of them. It can be hard to communicate with plants, even once you figure out their language. Take the plant abscisic acid (ABA) receptor, PYR1, for example. PYR1 communicates via ABA, a plant hormone that sounds the alarm for water stress and tells a plant to conserve water by closing its stomata.

Closing stomata helps plants keep water from escaping, but it also slows down their growth because CO2 can’t get in. Normally farmers want fast-growing crops with wide-open stomata, but when the weather forecast says no water for a while, it would be great if farmers could pass the word on to their crops. Unfortunately, ABA is too expensive to use every time there’s a dry spell.

Two years ago, the lab of Sean Cutler at UC Riverside discovered a new, potentially cheaper agonist, quinabactin, that could tell plants to hunker down for drought. However, quinabactin has yet to make it through the regulatory and commercialization gauntlet.

To find a quicker path to a practical drought signal, Cutler’s lab wondered if, instead of finding a new chemical that speaks PYR1’s language (i.e., an agonist), they could teach PYR1 to recognize a chemical that was already approved and available. Through a tour-de-force combination of screening, random mutagenesis, directed mutagenesis, and rational design – mostly affecting the receptor’s binding pocket – the team succeeded in making a new PYR1 variant that is deaf to ABA, but responds to mandipropamid instead. In one particularly striking experiment, mandipropamid was able to rescue arabidposis plants that otherwise would have died when left unwatered for 11 days, presumably during a lab retreat somewhere.

This approach to yelling “Drought!” to plants is a bit circuitous, of course. Farmers would have to be growing crops that had been engineered with the new receptor. Also, mandipropamid is a fungicide, and using it at every dry spell could help blight develop resistance to it.

However, the insight gained from this rewiring of the receptor could help the team teach it to recognize other chemicals too. In any case, this impressive bit of receptor re-engineering is definitely more effective than sitting out in the field with a guitar singing “It ain’t gonna rain no more no more”.

You can find the details on how plants have expanded their vocabulary with a new word for drought over in Nature, February 2015.

]]>http://epigenie.com/plant-receptor-re-engineered-to-hear-drought-from-a-new-signal/feed/0Expressed Long Non-coding RNAs Snitch on Tissue-specific Enhancershttp://epigenie.com/expressed-long-non-coding-rnas-snitch-on-tissue-specific-enhancers/
http://epigenie.com/expressed-long-non-coding-rnas-snitch-on-tissue-specific-enhancers/#respondWed, 11 Feb 2015 22:44:36 +0000http://epigenie.com/?p=22147Predicting tissue-specific enhancers is a tricky business. While histone marks such as H3K4me1 and binding of p300 are good enhancer predictors, pointing out which enhancers are tissue-specific is more of a challenge. That is, of course, unless you have PreSTIGE – an algorithm developed by the Scacheri lab that identifies tissue-specific enhancers by combining H3K4me1 and gene expression data.

Previously we have seen how long non-coding RNAs (lncRNAs) come in two flavors: those that are promoter associated and those that are enhancer associated. It is also known that long non-coding RNAs play a role in enhancer function and show highly tissue-specific expression. In the latest issue of Cell Cycle, the Orom and Scacheri labs put two and two together and find that having an overlapping lncRNA is actually a good indicator of enhancer tissue specificity. Using data from 11 human cell lines with different tissues of origin they show that:

]]>http://epigenie.com/expressed-long-non-coding-rnas-snitch-on-tissue-specific-enhancers/feed/0Stem Cells Take the Sting out of Radiotherapyhttp://epigenie.com/stem-cells-take-the-sting-out-of-radiotherapy/
http://epigenie.com/stem-cells-take-the-sting-out-of-radiotherapy/#respondWed, 11 Feb 2015 21:54:26 +0000http://epigenie.com/?p=22140Radiotherapy is an important and effective tool in the war against cancer, but its use for treating brain tumors brings with it unwanted side effects that can cause long term suffering to the patient. Radiation induces the depletion of oligodendrocyte progenitor cells (OPCs) which leads to the loss of the protective myelin sheath that covers neural cell axons causing cognitive decline and motor coordination impairment.

Up until now, there have been no successful treatments for these side effects, but researchers from the group of Viviane Tabar (Memorial Sloan Kettering Cancer) hope to change that. They have optimized a differentiation strategy for the production of replacement OPCs from human pluripotent stem cells (hPSCs) and show that upon transplantation into a rat model these OPCs have myelination capabilities that can reverse radiation induced side effects.

Oligodendrocytes produced from these cells all have the ability to efficiently myelinate axons in vitro.

After grafting hOPCs into the forebrain, extensive remyelination in the brain occurs, leading to improved performance in tasks reflecting multiple cognitive processes related to memory and learning.

Motor co-ordination improved after hOPC grafting into the cerebellum.

This is very welcome news to researchers, clinicians, and patients alike. A new protocol for the production of vast amounts of safe, clinically relevant patient-specific cells and a strategy to improve quality of life among cancer survivors has these researchers looking to the future. Not only do they hope for the implementation of a clinical trial and hopefully a first viable therapeutic option for radiotherapy-related side effects, but they also foresee the adaptation of their work into other fields where demyelination plays a major role in disease pathogenesis.

]]>http://epigenie.com/stem-cells-take-the-sting-out-of-radiotherapy/feed/0Culture Shock: The Transition to Transformed Cell Alters the Methylomehttp://epigenie.com/culture-shock-the-transition-to-transformed-cell-alters-the-methylome/
http://epigenie.com/culture-shock-the-transition-to-transformed-cell-alters-the-methylome/#respondTue, 10 Feb 2015 22:56:47 +0000http://epigenie.com/?p=22129Whether it’s making the monumental move to high school, switching jobs or experiencing the culture shock from moving countries, adjusting to a new environment isn’t always easy. Often we have to make concessions and tradeoffs, the extent of which is very much dependent on the type of environment we find ourselves in. It turns out that just like the unseasoned traveller, mammalian cells also need to make an adjustment to fit in when making the move to cell culture.

Recent findings have hinted epigenetic reprogramming in transformed culture cells; however, the degree and pace at which such gain or loss of DNA methylation occurs has not been fully deciphered.

Epigenetic modifications such as DNA methylation modulate chromosomal stability and gene expression without altering DNA sequence. Having previously discovered discrepancies in levels of 5’-hydroxymethylcytosine (5hmC) between transformed cells in culture and cells from the native tissue, Nestor and colleagues decided to explore how changes to the methylome occur during the transition from primary cell to transformed cell. To satisfy their curiosity, the team explored the effects of culture exposure on the methylome, hydroxymethylome, and transcriptome of mouse embryonic fibroblasts (MEFs) in a time-dependent fashion, using numerous 5hmC and 5’-methylcytosine (5mC) analysis techniques followed by expression microarray analysis.

Here is the summary of their findings on how mammalian cells get acquainted to culture systems:

Mammalian cells in culture undergo rapid global loss of 5hmC, but not 5mC.

Expression of Tet1 decreases upon cell exposure to culture.

Loss of 5hmC in culture occurs independent of 5mC levels.

Loss of demethylase activity leads to gain of 5mC, but not 5hmC at gene promoters.

Culture adaptation is characteristically marked with global redistribution of 5hmC.

“The implications of our work is that the Tet enzymes act as a part of a barrier against aberrant methylation in normal cells, at its most extreme the DNA modification changes that occur in cultured cells are linked to cellular transformation but do not necessarily drive it,” said Dr. Richard Meehan, the corresponding author of the study.